CASE REPORT (Olgu Sunumu)

A FALSE NEGATIVE QF-PCR AND TRISOMY 18-TRISOMY 9 MOSAICISM

Ebru DIKENSOY1, Ozcan BALAT1, Sacide PEHLIVAN2, Fatma Bahar CEBESOY1, Ali Irfan KUTLAR1, Tugce SEVER2, Volkan BALTACI3

1 Department of Obstetrics and Gynecology, Gaziantep University Faculty of Medicine, Gaziantep, Turkey 2 Department of Medical Biology and Genetics, Gaziantep University Faculty of Medicine, Gaziantep, Turkey 3 Gen-art Women’s Health an Reproductive Biotechnology Center, Ankara, Turkey

SUMMARY

Using QF-PCR for rapid prenatal diagnosis of major chromosome aneuploidies demonstrated that the method is highly efficient, reliable and cost effective in the literature. A discrepancy has been showed between QF-PCR and karyotyping results as 0.2% in mosaicism.Our case was in 17 th gestational week who referred to our clinic for having a trisomy 18 risk as 1 in 50 in triple test.On the ultrasonography, we detected bilateral choroid plexus cysts and bilateral pyelectasia.After amniocentesis QF-PCR has been showed a normal chromosomal patern and cytogenetic analysis has been showed trisomy 18 and trisomy 9 mosaicism.

Key words: mosaic trisomies, prenatal diagnosis, QF-PCR method Journal of Turkish Society of Obstetrics and Gynecology, (J Turk Soc Obstet Gynecol), 2011; Vol: 8 Issue: 1 Pages: 67- 70

ÖZET

YANLIfi NEGAT‹F QF-PCR VE TR‹ZOM‹ 18-TR‹ZOM‹ 9 MOZA‹ZM

Major kromozomal anöploidilerin h›zl› prenatal tan›s›nda Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR) yöntemi literatürde oldukça etkin (%95), güvenli ve düflük maliyetli olarak gösterilmifltir. Mozaik trizomilerde QF-PCR ve karyotipleme yöntemleri aras›nda % 0.2 oran›nda uyumsuzluk varl›¤› bildirilmifltir. Bizim olgumuz; 17. gebelik haftas›nda triple testinde trizomi 18 riskinin 1/50 olmas› nedeniyle klini¤imize gönderilmiflti. Yap›lan ultrasonografide fetusta bilateral koroid pleksus kistleri ve bilateral pelviektazi oldu¤u saptand›. Amniotik s›v›dan yap›lan QF-PCR analizi; normal kromozomal yap› gösterdi ancak sitogenetik analizde fetusta trizomi 18 ve trizomi 9 mozaizm oldu¤u tespit edildi.

Anahtar kelimeler: mozaik trizomiler, prenatal tan›, QF-PCR yöntemi Türk Jinekoloji ve Obstetrik Derne¤i Dergisi, (J Turk Soc Obstet Gynecol), 2011; Cilt: 8 Say›: 1 Sayfa: 67- 70

INTRODUCTION and painting operations following in-vitro culture that lasts for days is performed during the metaphase stage. Prenatal diagnosis of chromosomal abnormalities is Chromosome analysis performed with this analysis performed by cytogenetic analysis during the last four provides precise determination of all the numerical and decades, and during the last two decades they are structural abnormalities of the chromosomes(1). The diagnosed by Fluorescence In Situ Hybridization (FISH) main disadvantage is that the culture of fetal cells takes analysis. For the cytogenetic analysis appropriate banding a long time and the time elapses from taking the sample

Address for Correspondence: Dr. Ebru Dikensoy. Gaziantep Üniversitesi T›p Fakültesi, Kad›n Hastal›klar› ve Do¤um ABD, Gaziantep, Turkey Phone: + 90 (533) 496 20 77 e-mail: [email protected] Received: 12 August 2009, revised: 06 April 2010, accepted: 15 April 2010, online publication: 15 December 2010

67 DOI ID: 10.5505/tjod.2011.30306 Ebru Dikensoy et al. to receiving the result (an average of 14 days) might by the measurement of anterior-posterior diameter of cause anxiety in the family. This situation was shown the pelvis in the transverse cross-section of abdomen. to be more pronounced in patients that have an increased Because of the patient’s anxiety due to the possibility risk of chromosomal disorders according to non-invasive of trisomy and after she accepted the risk of abortion screening tests (biochemical and / or ultrasound) during due to amniocentesis, amniocentesis was performed. the first or second trimester(2). 48 hours following the amniocentesis QF-PCR result Quantitative Fluorescent Polymerase Chain Reaction performed by patented Gene Scan program using ABI (QF-PCR) analysis determines the major numerical 3100 revealed a normal chromosomal pattern (with 4 abnormalities (chromosomes 13,18,21, X and Y) within informative STR marker) (Figure 1). Because of the a few hours after the sample is taken, which is a method existence of two different ultrasonographic findings that had been used in the past 15 years(3). The analysis and high risk in the triple test result cytogenetic analysis of the trisomies in the amniotic fluid is based on was waited in order to reach a definite decision. On reproduction of the chromosome specific small DNA the 15th day of amniocentesis mosaic structure of sequences (STR markers containing 3,4,5 repeats) and Trisomy 18+Trisomy 9 was detected (Figure 2,3). capillary electrophoresis analysis. While using selected Proportion of abnormal cells in the karyotype was 10% chromosome-specific 17 STR markers they are diagnosed in total and the structure of 86% of these trisomic cells according to 1.2 or 3 peaks they create and with the was 47XY +18, and 14% of them were 47 XY+9(10). analysis of gap between size and space with a special Amniotic cells were inspected in 2 separate flasks. In program(1,4-6). This method, which was first used by the first flask 50 cells were inspected; 44 of them were Mansfield in 1993 in the analysis of Down syndrome 47, XY +18 and 6 of them were 47, XY +9. In the and other aneuploids, is being used in the routine check second flask 20 cells were inspected; 16 of the were after analyzing thousands of different populations. In found as 47, XY +18 and 4 of them were 47, XX+9. the Turkish population in 2007 Onay et al compared This situation was thought to be compatible with 3rd both the QF-PCR and cytogenetic results of the amniotic grade mosaicism. The patient was told that fluid and found similar results to other populations(5). cordocentesis should be performed for a definitive In a study presented by Ozkinay et al in 2008 the results diagnosis, but she did not accept this process. The of a 3-year audit was shown to be compatible with the patient was informed about the prognosis of mosaicism, literature(7). However, mosaicisms that have a low rate however, the pregnancy continued since she did not (less than 10%) might not be recognized(8,9). In this accept termination of the pregnancy. study, we presented a case that could not be diagnosed In the ultrasonography performed during the 21st week with QF-PCR following amniocentesis but trisomy 18 of pregnancy, an expansion in the posterior fossa of the and trisomy 9 mosaicism detected with karyotyping. fetus was observed (foramen magnum was measured as 12 mm). Bilateral choroid plexus cysts and pyelectasis were persisting in the same sizes. During the follow-up CASE REPORT pregnancy continued without problems until term. The patient had a vaginal delivery in a special center and the 23-year-old17-week pregnant woman gravida 2, parity baby was ex postpartum at the 4th hour. 1 was referred to our clinic for amniocentesis because the risk of trisomy 18 in the triple test was reported as 1/50. In the triple test, serum alpha-fetoprotein was 27.96 IU / ml (0.81 MOM), uE3 1 ng / ml (0.80 MOM) and hCG 18,213 mIU / ml (0.46 MOM) and the risk of trisomy 18 was calculated as 1/50. The detailed obstetric ultrasound revealed bilateral pyelectasis and bilateral choroid plexus cysts in the fetus. The choroid plexus cyst was measures as 6 mm in the right and 5 mm in the left. The presence of pyelectasis was Figure 1: QF-PCR analysis of chromosones 13,18 and the sex chromosomes. determined 7 mm in the right and 5.9 mm in the left *There is no maternal contamination

J Turk Soc Obstet Gynecol 2011; 8: 67- 70 68 A false negative QF-PCR and trisomy 18-trisomy 9 mosaicism

nucleotides. DNA is replicated by using fluorescent primers of PCR and this product could be seen with a gene browser program as each length repeated using automated DNA sequencer might be seen as the peak area and are comparable(8,9). 65-95% of the DNA replicated from the normal people is heterozygote (includes different lengths of alleles) and are expected to have two peaks in the same area(7). DNA replicated from the trisomic people;might reveal and extra peak in the same regions in the triallelics and only two peaks in Figure 2: Metaphase plates, trisomy of 18th chromosome is shown the diallelics and one might be twice as long as the with G banding. other(7). Because QF-PCR is a method that works with duplication of DNA and isolated DNA was amplified at least for 20 cycles it fails show a difference in the DNA below 20%(1-5). This method can also be applied to different fetal tissues (amniotic fluid, fetal blood, chorionic villi and fetal tissues after termination) and might show us the maternal contamination with blood samples taken from the mother. result can be given by working with at least 2 informative markers for each of the chromosome number(13-18). In our case, DNA markers were informative and the patient had intermarriage. Figure 3: Trisomy 18 is seen in karyotyping. FISH and QF-PCR quickly show whether the examined chromosomes are aneuploidy or not (24-48 hours). Both methods might be used in the recognition of all the DISCUSSION chromosomes, but they were developed for the routine use of 13, 18, 21 chromosomes and sex chromosomes. In the early 1990s fluorescence in situ hybridization These methods could be used for the high-risk patients (FISH) and very recently QF-PCR are the two methods (fetal malformation / soft marker are determined), or used for prenatal diagnosis in order to address the need for the patients in the advanced weeks of pregnancy that of fetal cell culture. They provide rapid diagnosis of are late for cytogenetic analysis. They are reliable to some specific chromosomal abnormalities(1-10). FISH intervene when a chromosomal abnormality is detected, is being performed by using specific labeled DNA but some geneticists might accept them as a start while probes that are detectable using chemically modified waiting for the result of karyotyping. In fact, the and directly emitting fluorescence or could be fixed chromosome disorders studied with these rapid survey by a connecting molecule emitting fluorescent(5-11). techniques consist only 65% of changes observed in Normally, when the nuclei of fetal cells are analyzed high-risk population(14). QF-PCR method contains some with fluorescent microscopy analysis two points would advantages compared to the FISH method. QF-PCR be observed for each one of the chromosomes that is analysis provides examinateion of a much larger number examined. Trisomies have an extra point, and of cells compared to FISH. Since this process could be monosomies lack one point(6-12). easily automated numerous samples could be studied at The most commonly used form of QF-PCR is the same time and the whole process takes only 30 chromosome-specific, containing replications of repeats minutes. In the FISH method there are specific probes of small 3.4, or 5 nucleotide DNA in the repetitive DNA and they do not require any authentication(9-14). While sequences. Repeated short DNA sequences are QF-PCR might define maternal cell contamination, standardized and polymorphic, and the length of them FISH might not revealthis for the female fetuses. For varies significantly among the people like a fingerprint these reasons, QF-PCR is being rapidly accepted for the according to repeated triple, quadruple or quintet prenatal diagnosis as an alternative to conventional

69 J Turk Soc Obstet Gynecol 2011; 8: 67- 70 Ebru Dikensoy et al. cytogenetic analysis(10-16). Like some other prenatal Perez MM, Fuster C, Egozcue J. Clinical application of multiplex diagnostic tests QF-PCR cannot define the mosaicism quantitative polymerase chain reaction (QF-PCR) fort he rapid with a low rate. Low mosaicism rates (less than 10%) prenatal detection of common chromosome aneuploides. Mol could not be defined by molecular testing(4-8). The ability Hum Reprod 2001; 7: 1001- 6. of QF-PCR in sensitivity of defining two or more cell 5. Onay H, Ugurlu T, Aykut A, Pehlivan S, Inal M, Tinar S, Ozkinay lines the mosaic patients depends on the rate of mosaic C, Ozkinay F. Rapid Prenatal Diagnosis of Common Aneuploidies cells and the involved chromosomes. For example, 46, in Amniotic Fluid Using Quantitative Fluorescent Polymerase XX/45,X mosaics could be diagnosed with an unbalanced Chain Reaction. Gynecol Obstet Invest 2008; 66: 104- 10. ratio using X chromosome markers, when at least 20% 6. Nakonieczny M, Jezierski G, Wozniak I, Liss J, Pawlowski of cells of aneuploids are seen. In some mosaic fetuses R, Preis K, Swiatkowska-Freund M, Wójcikowski C, Lukaszuk containing 46, XX/45,X or 45,X/47XXX or 46, XX/45, K. Prenatal diagnosis of fetal chromosomal aneuploidies using quantitative fluorescent PCR (QF-PCR)]. Przegl Lek. 2008; X/47, XXX, determination the type and rate of different 65(3): 119- 21. Polish. cell groups in fetuses is quite difficult. Because they 7. Ozkinay F, Pehlivan S, Alpman A, Onay H, Cogulu O, Ak›n can create fluorescent peaks like the normal 46, XX or H, Sagol S, Akercan F, Kazandi M, Balat O, Kutlar I, Ozkinay 47, XXX(17,18). In some cases, the results of QF-PCR C. Prenatal Detection of common aneuploidies by quantitative and cytogenetic analysis in X chromosome mosaicism fluorescence PCR in The Turkish Population: Three years discrepancies might occur depending on the ratio of experience in two reference hospitals. XXI. European Congress abnormal small cell population. This discrepancy rate of Perinatal Medicine, Istanbul-Turkey, The Journal of Maternal- (1) has been reported as 0.2% in the literature . This is Fetal & Neonatal Medicine, (Supplement 1), 2008; 55: 21. because of the differences in the in vitro growth rate of 8. Sherlock J, Cirigliano V, Petrou M, Tutscheck B, Adinolfi normal (46, XX or 46, XY) and 45,X cells. Aneuploid M. Assesment of diagnostic quantitative fluorescent multiplex cells grow faster than the normal cells. However, in the chain reaction assays peformed on single cells. Ann Hum presence of more than one cell line QF-PCR allows Genet 1998; 62: 9- 23. diagnosis in the 50% of the mosaic trisomies that were 9. Lau ET, Tang L, et al. Assessing discrepant findings between diagnosed with cytogenetic analysis(1). QF-PCR on uncultured prenatal samples and karyotyping on long-term culture. Prenatal Diagn 2009; 29: 151- 5. In conclusion, in the literature when QF-PCR method 10. Chia NL. A Comprehensive Set of Idiograms Representing is used alone as the standard method it might miss the All Interpretive Levels of Resolution: ISCN (2009). Cytogenet single chromosome abnormalities that have an increased Genome Res 2009; 125: 162- 4 (DOI: 10.1159/000227842). frequency with the advanced age according to distribution 11. Von Eggeling F, Freytag M, Fahsold R, Horsthemke B, Claussen of age. The rate of this is 1 in every 150 abnormal U. Rapid Detection of trysomy 21 by Quantitative PCR. Hum (13-18) karyotype and one in every 10-30000 . Even though, genet 1993; 91 567- 70. it might miss some sex chromosome abnormalities, 12. Hulten MA, Dhanjal S, Pertl B. Rapid and prenatal diagnosis mostly structural, in the literature it has been emphasized of common chromosome disorders: advantages and disadvantages that this error rate is acceptable(8-13). of the molecular methods FISH and QF-PCR. Reproduction 2003; 126: 279- 97. 13. Grimshaw GM, Szczepura A, Hulten M, MacDonald F, Nevin NC, Sutton F, et al. Evaluation of molecular tests for prenatal REFERENCES diagnosis of chromosomes abnormalities. Health Technology Assesment 2003; 7: 1- 56. 1. Mansfield ES. Diagnosis of Down syndrome and other aneuploides 14. Winsor EJ, Silver MP, Theve R, Wright M, Ward BE. Maternal using quantitative polymerase chain reaction and small tandem cell contamination in uncultured amniotic fluid. Prenatal repeat polymorphisms. Mol Hum Genet 1993; 2: 43- 50. diagnosis 1996; 16: 49- 54. 2. Cirigliano V, Voglino G, et al. Rapid prenatal diagnosis of 15. Cirigliano V, Sherlock J, Conway G, Quilter C, Rodeck C, common chromosome aneuploidies by QF-PCR, results of 9 Adinolfi M. Rapid detection of chromosomes X and Y aneuploides years of clinical experience. Prenatal Diagn 2009; 29: 40- 9. by quantitative fluorescent PCR. Prenat Diagnosis 1999; 19: 3. Sjogren B, Uddenberg N. Prenatal diagnosis for psychological 1099- 103. reasons: comparison with other indications, advanced maternal 16. Schmidt W, Jenderny J, Hecher K, et al. Detection of aneuploidy age and known genetic risk. Prenat Diagn 1990; 10: 111- 20. in chromosomes X,Y, 13,18 and 21 by QF-PCR in 662 selected 4. Cirigliano V, Ejargue M, Carradas MP, Lloveras E, Plaja A, pregnancies at risk. Mol Hum Reprod 2000; 6: 855- 60.

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